Objective 1: Develop a robust, scalable and clinically-relevant culture system for hematopoietic differentiation of human iPSCs. To facilitate the development of functional HSCs from human iPSCs, we developed a simple, monolayer-based, chemically-defined, and scalable differentiation protocol requiring no replating or embryoid body (EB) formation (commercially available as STEMdiffTM Hematopoietic Kit, Stem Cell Technologies). Human iPSCs reprogrammed from CD34+CD38- cells of a healthy volunteer (MCND-TENS2) were subjected to hematopoietic differentiation for 21 days using this approach. Under culture conditions that favored mesodermal specification (Day 0 to 3), an adherent monolayer rapidly formed. With the subsequent addition of hematopoietic cytokines (Day 3 to 21), hematopoietic clusters emerged from the monolayer before their eventual release in the supernatant fraction. We systematically characterized cells arising from this system by harvesting supernatant and monolayer populations at regular intervals between day 5 and 21 of differentiation. Hematopoietic cells were characterized by varying expression of CD43, the earliest marker of human hematopoietic commitment, and by gradual acquisition of the pan-hematopoietic marker CD45 (CD43+CD45+/-). In contrast, non-hematopoietic cells formed a distinct population expressing neither markers (CD43-CD45-). 1.1 Characterization of the hematopoietic fraction To determine whether hematopoietic differentiation of human iPSCs using this system can provide a suitable ex vivo model to study the emergence of hematopoiesis, we characterized the hematopoietic CD43+CD45+/- fraction throughout differentiation by flow cytometry and functional assays. Our data indicate that hematopoietic differentiation of human iPSCs with STEMdiffTM enables the sequential development of hematopoietic cells with features of primitive wave one-hematopoiesis (peak at day 7), definitive multilineage HSPCs with potent colony formation activity in vitro but limited engraftment potential in vivo (peak at day 12), and definitive erythroid-committed progenitors expressing adult-type globin chains (peak at day 17 to 21). This nearly exclusive shift to definitive erythroid growth in later stages of differentiation could be exploited to facilitate erythroid differentiation of iPSCs established from patients with hemoglobinopathies or various congenital bone marrow failures and anemias affecting early erythropoiesis. The robust generation of patient-specific erythroid progenitors could have considerable implications, including ex vivo modeling of disease pathophysiology, preclinical screening of gene therapy strategies, and specific testing of novel therapeutics against disease-relevant human cells. 1.2 Characterization of the non-hematopoietic fraction To understand the possible causes underpinning the absence of engraftable HSCs in this system, we examined the cellular constituents of the supportive non-hematopoietic niche. We first identified a prevalent population of phenotypically defined mesenchymal cells throughout differentiation. Perivascular mesenchymal cells are known to interact with HSCs and maintain their activity in the adult BM niche. However, their role in promoting HSC development during ontogeny has not been demonstrated. Instead, mesenchymal cells have been implicated as components of the niche in the yolk sac controlling primitive erythroid cell maturation. Further investigation is needed to fully understand whether these cells may offer inhibitory signals that preclude normal developmental switch to third wave definitive hematopoiesis during iPSC differentiation. We also found limited vascular endothelium (VE) production and arterial specification within the non-hematopoietic fraction that may account for the lack of engrafting HSCs in culture. Indeed, recent advances propose that formation of these cells is restricted to arterial vessels during ontogeny. Thus, in keeping with this model, arterial VE are likely inadequate to support the generation of bona fide engrafting HSCs in culture. Ongoing single cell RNA sequencing (RNA-seq) and ATAC-seq studies will elucidate differences in gene expression and chromatin accessibility between bona fide and iPSC-derived HSPCs, and inform possible strategies to overcome the engraftment deficit. 1.3 Impact of CHIR/SB of hematopoietic and non-hematopoietic development Our study also provides proof-of-principle that this monolayer iPSC differentiation system is readily amenable to simple, clinically applicable modifications to improve hematopoietic output. Quantitative modulation of WNT/beta-catenin and activin/nodal/TGF signaling pathways by one-time addition of CHIR/SB molecules during mesodermal specification enhanced arterial VE and increased HOXA gene expression and definitive HSC formation. However, consistent with previous studies, this approach alone was insufficient to orchestrate the formation of functional HSCs and additional revisions to this system will be required. This work in under review (Stem Cell Research 2019). Objective 2: Uncover and activate human HSC-specific super-enhancers (SE) for the conversion of iPSCs into functional HSCs. The CRISPR-Cas9 system offers the opportunity to precisely manipulate genomic loci specified by a guide RNA (gRNA). An adaptation of the CRISPR technology has recently emerged whereby nuclease-deactivated Cas9 (dCas9) is fused to transcriptional activator (e.g. VP64) or epigenome modulator (e.g. p300) domains to precisely upregulate gene expression from endogenous promoters and both proximal and distal enhancer regions. Importantly, Cas9 can be used with pooled gRNA libraries for high-throughput CRISPR-activation (CRISPRa) screens. We are developing this technology for the targeted activation of HSC-specific genes driven by SE (see goals above) as a novel alternative approach for the conversion of iPSCs into transplantable HSCs. Work is ongoing in three specific aims: 2.1 Identification of HSC-specific SE SE are identified by bioinformatic search for clusters of enhancer-associated surrogate epigenetic marks such as H3K27ac, H3K4Me1 or DNase I hypersensitivity (HS) sites, and by the absence of histone marks associated with promoter function (H3K4me3), repressor activity (H3K27me3) or heterochromatin state (H3K9me3). We are investigating SE-histone marks by CHIP-seq, and SE-DNase HS sites by ATAC-seq on HSC-enriched CD34+CD38- cells derived from healthy individuals. 2.2 Activation of HSC-specific gene expression We are developing an inducible lentiviral vector-based high-throughput CRISPRa approach to activate HSC-specific gene expression in human iPSCs by: 1) Targeting the promoter regions of HSC-specific master TFs with CRISPR-dCas9VP64; 2) Targeting locations of DNase HS sites or locations of activating H3K27Ac histone marks in HSC-specific SE with CRISPR-dCas9p300. 2.3 Characterization of the impact of HSC-specific gene activation in iPSCs. Cells derived following CRISPRa-mediated activation of HSC-specific genes in iPSCs are characterized by: 1) Phenotypic analysis (morphology, flow cytometry); 2) Molecular analysis (RNA-seq, epigenomic); 3) Functional analysis (CFU assay, NSG mouse transplantation).